Radioactive proteins synthesized in a cell-free system under the direction of mRNA from A+T-20-D16v cells were isolated by immunoprecipitation with antiserum to ACTH or beta-endorphin. Each immunoprecipitate was shown to contain a single radioactive protein with an apparent molecular weight of 28,500. These proteins, which had the same tryptic peptide maps, also contained the sequences of both alpha (1-39) ACTH and beta-LPH. Beta-LPH was shown to be located C-terminal to ACTH in the cell-free product. The cell-free product and cyanogen bromide fragments from the cell-free product are being used for amino acid sequence determination by an automatic Edman degradation procedure. The ACTH-LPH messenger RNA was purified and used as a template to make complementary DNA (cDNA). The cDNA was digested with specific bacterial restriction endonucleases and the restriction fragments were purified by gel electrophoresis for nucleotide sequence analysis. Pulse chase studies with radioactive amino acids and sugars in the cell line suggest that different processing pathways are involved in the conversion of the ACTH-LPH precursor to 13 K ACTH and 4.5 K ACTH (13K ACTH is the glycosylated form of 4.5 K ACTH). Tumor cultures and primary pituitary cell cultures were shown to release beta-LPH in addition to 13K and 4.5K ACTH. Regulation of the release of all of these hormones by hypothalamic extract and dexamethasones is being studied.